The present invention relates generally to recombinant plant viral nucleic acids, and methods of use thereof.
Recombinant plant viral nucleic acids are of interest generally for their utility as expression vectors in plants.
Additionally, such nucleic acids can be used to initiate virus induced gene silencing (VIGS). This phenomenon is based on the observation that virus infection in plants can initiate sequence-specific nucleic-acid based defence mechanisms that resemble either transcriptional, or post-transcriptional gene silencing (PTGS) (Covey, Al-Kaff 1997; Ratcliff, Harrison et al. 1997; Al-Kaff, Covey et al. 1998). PTGS is also manifest as an inhibition of nuclear gene expression when a virus is modified to carry sequence from a nuclear expressed gene (Kumagai, Donson et al. 1995; Kjemtrup, Sampson et al. 1998; Ruiz, Voinnet et al. 1998). PTGS can also cause recovery from viral infection when a plant expressing a transgene derived from viral cDNA is infected by a homologous virus (Lindbo, Silva-Rosales et al. 1993; Guo and Garcia 1997). Both the inhibition of nuclear gene expression, and recovery from viral infection are caused by sequence-specific RNA degradation.
Because modified viruses inhibit the expression of homologous plant genes, VIGS can be used to induce an apparent null-phenotype or a loss of function and therefore identify the function of any gene. Viruses that have been modified in this manner include tobacco mosaic virus (TMV) (Kumagai, Donson et al. 1995) potato virus X (PVX) (Ruiz, Voinnet et al. 1998), and tomato golden mosaic virus (Kjemtrup, Sampson et al. 1998).
The present invention is concerned with novel recombinant plant viral nucleic acids.
In preferred forms the present invention is concerned with providing VIGS-based methods and materials which may be more suitable as a tool for functional genomics than those which have been used in the past. For instance TMV, PVX and TGMV infections cause significant symptoms, such as a chlorosis, leaf-distortion and necrosis. Phenotypes caused by VIGS of a plant gene can therefore be hard to differentiate from these viral symptoms. Secondly, like most viruses, TMV, PVX and TGMV form mosaic, vein-based infections, and therefore do not cause confluent VIGS across the whole leaf. Leaves may therefore contain a mixture of cells with and without VIGS, complicating interpretation of any phenotype. Thirdly, TMV, PVX and TGMV do not infect meristems (Matthews 1991) and can not therefore inhibit expression of genes that determine the identity and development of plant tissue. Finally, although the first plant genome to be fully sequenced will be that of Arabidopsis thaliana, TMV, PVX and TGMV vectors do not infect this plant. Therefore the potential of VIGS to identify gene function in Arabidopsis is limited with available technology. A VIGS vector which overcame one or more of these drawbacks would therefore represent a contribution to the art.
The present inventors have developed novel recombinant cDNA viral constructs based on tobacco rattle virus (TRV) which in preferred forms are particularly adapted for use with VIGS. Such vectors may induce few or no symptoms, cause confluent VIGS across the leaf, operate in Nicotiana species and in Arabidopsis, and inhibit gene expression in meristems.
A viral expression vector based on TRV has previously been described in which non-viral proteins were expressed from a sub-genomic promoter (Ratcliff, MacFarlane et al. 1999). The viral RNA was synthesised in vitro and then inoculated into the plant. The TRV vectors of the present invention include inter alia modifications to facilitate both the insertion of plant gene sequences and the it subsequent infection of plants. Other TRV based vectors are disclosed by Hamilton and Baulcombe (1989) J. Gen. Virol 70: 963-968 and Mueller et al (1997) J. Gen. Virol 78: 2085-2088.
Thus in a first aspect of the present invention there is disclosed a nucleic acid vector which comprises:
(a) a transfer nucleotide sequence comprising (i) a plant active promoter, operably linked to (ii) a recombinant tobacco rattle virus (TRV) cDNA which includes at least cis acting elements permitting replication of said cDNA; a subgenomic promoter operably linked to a sequence encoding a TRV coat protein; and a heterologous nucleotide sequence which is foreign to said virus;
(b) border sequences which permit the transfer of the transfer nucleotide sequence into a plant cell nucleus.
The transfer nucleotide sequence is situated between the border sequences and is capable of being inserted into a plant genome under appropriate conditions. Generally this may be achieved by use of so called xe2x80x9cagro-infiltrationxe2x80x9d which uses Agrobacterium-mediated transient transformation. Briefly, this technique is based on the property of Agrobacterium tumafaciens to transfer a portion of its DNA (xe2x80x9cT-DNAxe2x80x9d) into a host cell where it may become integrated into nuclear DNA. The T-DNA is defined by left and right border sequences which are around 25 nucleotides in length. In the present invention the border sequences are included around the transfer nucleotide sequence (the T-DNA) with the whole vector being introduced into the plant by agro-infiltration, optionally in the form of a binary-transformation vector.
By xe2x80x9cplant active promoterxe2x80x9d is meant a sequence of nucleotides from which transcription may be initiated of DNA operably linked downstream (i.e. in the 3xe2x80x2 direction on the sense strand of double-stranded DNA). xe2x80x9cOperably linkedxe2x80x9d means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter. Nucleic acid operably linked to a promoter is xe2x80x9cunder transcriptional initiation regulationxe2x80x9d of the promoter.
The cDNA includes cis acting elements permitting replication of said cDNA. However the vector need not include all of the sequence required to replicate and move within the plant. The vectors of the present invention will generally require supplementary proteins and/or nucleic acids from TRV in order to achieve this. Thus the cDNA may correspond to part of TRV RNA 2, and will thus require proteins encoded by TRV RNA1 for replication.
The TRV coat protein (as with other defined or recited sequences herein) need not be xe2x80x98wild-typexe2x80x99, but may optionally be a variant (e.g. mutant, or other variant, or a substantially homologous derivative) provided that its function (to encapsulate and permit movement of the TRV genome) is not negated. By xe2x80x9cSubstantially homologousxe2x80x9d is meant that the sequence in question shares at least about 70%, or 80% identity, most preferably at least about 90%, 95%, 96%, 97%, 98% or 99% identity with the reference sequence. Identity may be at the nucleotide sequence and/or encoded amino acid sequence level. Homology may be over the full-length of the relevant sequence shown herein (e.g. in the sequence Annex) or may be over a part of it. Identity may be determined by the TBLASTN program, of Altschul et al. (1990) J. Mol. Biol. 215: 403-10, or BestFit, which is part of the Wisconsin Package, Version 8, September 1994, (Genetics Computer Group, 575 Science Drive, Madison, Wis., USA, Wisconsin 53711). Preferably sequence comparisons are made using FASTA and FASTP (see Pearson and Lipman, 1988. Methods in Enzymology 183: 63-98). Parameters are preferably set, using the default matrix, as follows: Gapopen (penalty for the first residue in a gap): xe2x88x9212 for proteins/xe2x88x9216 for DNA; Gapext (penalty for additional residues in a gap): xe2x88x922 for proteins/xe2x88x924 for DNA; KTUP word length: 2 for proteins/6 for DNA.
The heterologous nucleotide sequence is foreign (non-native) to TRV, which is to say that it does not occur naturally in the TRV viral genome at the position in which it is present in the VIGS vector. The sequence will generally be either a cloning site (to permit the insertion of a desired sequence) or a desired sequence itself.
Some preferred embodiments of the invention will now be discussed.
Vector
This is preferably based on plant binary transformation vector pGreen (see Materials and Methods below). The vector may be an expression vector (for transcription of a desired sequence, which may then be translated). Alternatively (and preferably) the vector is a xe2x80x9cVIGS vectorxe2x80x9d, by which is meant one which is adapted to cause or permit virus induced gene silencing of a desired target nucleotide sequence corresponding to a sequence included in the vector.
Nucleic acid vectors according to the present invention may be provided isolated and/or purified, in substantially pure or homogeneous form, or free or substantially free of other nucleic acid. The term xe2x80x9cisolatedxe2x80x9d encompasses all these possibilities.
Generally speaking, in the light of the present disclosure, those skilled in the art will be able to construct vectors according to the present invention. Such vectors may include, in addition to the promoter, a suitable terminator or other regulatory sequence such as to define an expression cassette consisting of the recombinant TRV cDNA and the heterologous nucleotide sequence. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al, 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley and Sons, 1992. Specific procedures and vectors previously used with wide success upon plants are described by Bevan, Nucl. Acids Res. (1984) 12, 8711-8721), and Guerineau and Mullineaux, (1993) Plant transformation and expression vectors. In: Plant Molecular Biology Labfax (Croy RRD ed) Oxford, BIOS Scientific Publishers, pp 121-148.
Plant Promoter
Suitable promoters will be well known to those skilled in the art and include the Cauliflower Mosaic Virus 35S (CaMV 35S) gene promoter that is expressed at a high level in virtually all plant tissues. The promoter may in principle be an inducible promoter such as the maize glutathione-S-transferase isoform II (GST-II-27) gene promoter which is activated in response to application of exogenous safener (WO93/01294, ICI Ltd). The GST-II-27 gene promoter has been shown to be induced by certain chemical compounds which can be applied to growing plants. Another suitable promoter may be the DEX promoter (Plant Journal (1997) 11: 605-612).
Recombinant TRV cDNA
This is preferably based on a modified, reduced, cDNA clone of TRV RNA2. In the Examples herein the strain used is ppk20. However any appropriate strain, which can give rise to replicating, infectious viral transcripts, could be used (see e.g. Macfarlane, 1999 for further examples).
Within the cDNA it is preferable that non-essential ORFs or other sequences are deleted, provided that the cDNA can still be used to generate replicating, infectious transcripts. Preferably, where the cDNA is based on TRV RNA2 of ppk20, two open reading frames (37K and 32.8K) are deleted to leave only the 5xe2x80x2 and 3xe2x80x2 untranslated regions and the viral gene encoding the coat-protein. The deleted ORFs are replaced by a heterologous nucleotide sequence between the coat protein and the untranslated region (UTR). The sequence is shown in the Sequence appendix (No. 1). Naturally substantially homologous variants of the sequence are also included within the scope of the invention. In particular, vectors derived from pTV00 and having the characteristics (described herein) of that vector, are also embraced.
Vectors based on TRV RNA2 require proteins encoded by TRV RNA1 for replication, which can be achieved as described below.
Heterologous Nucleotide Sequence.
This can in principle be a single or multiple cloning site (i.e. a sequence encoding two or more restriction endonuclease target sites) to facilitate the incorporation of a desired nucleotide sequence.
For expression vectors according to the present invention, the sequence will generally include or be operably linked to a subgenomic promoter which is recognised by a TRV-effective replicase (e.g. the PEBV CP subgenomic promoter) and an ORF sequence which it is desired to express and which is therefore transcribed as a subgenomic RNA.
For VIGS vectors the sequence will be a xe2x80x9ctargeting sequencexe2x80x9d which corresponds to a sequence in a target gene, either in the sense or anti-sense orientation, or a sequence which has sufficient homology to a target sequence for down-regulation of expression of the target gene to occur. Such a targetting sequence may be included in the vector anywhere in the viral cDNA irrespective of the location of any subgenomic promoter (provided it does not interfere with the cis-acting replication elements or the coat protein). Generally speaking it will be preferable for VIGS vectors according to the present invention not to include a subgenomic promoter within or operably linked to the heterologous gene sequence. Such preferred vectors have the advantage that they are more stable (reduced likelihood of self-recombination) that those of the prior art such as those described by Ratcliff, MacFarlane et al. (1999) supra which had more than one subgenomic promoter.
In general the targeting sequence may be derived from a plant nuclear gene or transgene, or a gene on an extrachromosomal element such as a plastid.
VIGS is particularly preferred for investigating gene function in that it can be used to impose an intermediate or a null phenotype for a particular gene, which can provide information about the function of that gene in vivo. In such cases the targeting sequence may not be known, but the methods of the present invention may be used to identify it with a particular phenotype.
The complete sequence corresponding to the coding sequence (in reverse orientation for anti-sense) need not be used. For example fragments of sufficient length may be used. It is a routine matter for the person skilled in the art to screen fragments of various sizes and from various parts of the coding sequence to optimise the relationship between target and targeting sequence. It may be preferable that there is complete sequence identity between the targeting sequence in the vector and the target sequence in the plant, although total complementarity or similarity of sequence is not essential. One or more nucleotides may differ in the targeting sequence from the target gene. Thus, a targeting sequence employed in a construct in accordance with the present invention may be a wild-type sequence (e.g. gene) selected from those available, or a substantially homologous mutant, derivative, variant or allele, by way of insertion, addition, deletion or substitution of one or more nucleotides, of such a sequence. Such a sequence need not include an open reading frame or specify an RNA that would be translatable.
A further possibility is to target a conserved sequence of a gene, e.g. a sequence that is characteristic of one or more genes in one or more pathogens against which resistance is desired, such as a regulatory sequence.
Other aspects of the invention will now be discussed.
One aspect of the present invention is a process for producing a vector as described above, the process being substantially as set out in the Examples hereinafter. A further aspect is a process for producing a vector as described above, which process comprises the step of cloning a heterologous nucleotide sequence which is a targeting sequence into the vector.
Thus one aspect of the present invention includes a method of silencing a target gene in a plant tissue using VIGS which method comprises the steps of introducing a vector as described above into the plant, wherein said vector includes a heterologous nucleotide sequence which is a targeting sequence.
xe2x80x9cPlant tissuexe2x80x9d is any tissue of a plant in planta or in culture, including the whole plant an organ thereof, a cutting, or any group of plant cells organised into a structural and functional unit.
xe2x80x9cSilencingxe2x80x9d is a term generally used to refer to suppression of expression of a gene. The degree of reduction may be so as to totally abolish production of the encoded gene product, but more usually the abolition of expression is partial, with some degree of expression remaining. The term should not therefore be taken to require complete xe2x80x9csilencingxe2x80x9d of expression. It is used herein where convenient because those skilled in the art well understand this.
The method may be preferably used to cause confluent VIGS of the target gene across a whole leaf and/or to silence a target gene in meristematic tissue.
As discussed above, for introduction into the plant, the vector may be in the form of an Agrobacterium binary vector. The vector is introduced into the plant cell by Agrobacterium-mediated T-DNA transfer, the transfer sequence may be integrated transiently into the plant (cell) genome, and is then transcribed to RNA from the plant promoter. In the published vector of Ratcliff, MacFarlane et al. (1999), the viral cDNA and any cDNA inserted after the sub-genomic promoter was transcribed to infectious RNA in vitro by T7 RNA polymerase and subsequently introduced into the plant.
TRV RNA 2 and all derived constructs require proteins encoded by TRV RNA1 for replication within and movement though out the plant. TRV RNA1 infections can be initiated either by rub-inoculating the plant with purified RNA 1 (Matthews 1991), or by transient Agrobacterium mediated expression in the plant of the plasmid pBINTRA6, which contains a CaMV 35S driven infectious clone of TRV PPK20 RNA 1 (see Materials and Methods).
The present invention may particularly be applied in plants which are natural hosts (compatible with) TRV. By xe2x80x9ccompatiblexe2x80x9d is meant capable of operating with the other components of a system, in this case TRV must be capable of replicating in the plant in question. These include Arabidopsis thaliana. Others include (but are not limited to) Allium cepa; Amaranthus caudatus; Amaranthus retroflexus; Antirrhinum majus; snap-dragon; Arachis hypogaea; Avena sativa; Bellis perennis; Beta vulgaris; Brassica campestris; Brassica campestris ssp. napus; Brassica campestris ssp. pekinensis; Brassica juncea; Calendula officinalis; Capsella bursa-pastoris; Capsicum annuum; Catharanthus roseus; Cheiranthus cheiri; Chenopodium album; Chenopodium amaranticolor; Chenopodium foetidum; Chenopodium quinoa; Coriandrum sativum; Cucumis melo; Cucumis sativus; Glycine max; Gomphrena globosa; Gypsophila elegans; Helianthus annuus; Hyacinthus; Hyoscyamus niger; Lactuca sativa; Lathyrus odoratus; Linum usitatissimum; Lobelia erinus; Lupinus mutabilis; Lycopersicon esculentum; Lycopersicon pimpinellifolium; Melilotus albus; Momordica balsamina; Myosotis sylvatica; Narcissus pseudonarcissus; Nicandra physalodes; Nicotiana benthamiana; Nicotiana clevelandii; Nicotiana glutinosa; Nicotiana rustica; Nicotiana sylvestris; Nicotiana tabacum; Nicotiana edwardsonii; Ocimum basilicum; Petunia hybrida; Phaseolus vulgaris; Phytolacca americana; Pisum sativum; Raphanus sativus; Ricinus communis; Salvia splendens; Senecio vulgaris; Solanum melongena; Solanum nigrum; Solanum tuberosum; Spinacia oleracea; Stellaria media; Trifolium pratense; Trifolium repens; Tropaeolum majus; Tulipa; Vicia faba; Vicia villosa; Viola arvensis. 
Target genes include those which confer xe2x80x98unwantedxe2x80x99 traits in the plant and which it may therefore be desired to silence using VIGS. Examples include ripening specific genes in tomato to improve processing and handling characteristics of the harvested fruit; genes involved in pollen formation so that breeders can reproducibly generate male sterile plants for the production of F1 hybrids; genes involved in lignin biosynthesis to improve the quality of paper pulp made from vegetative tissue of the plant; gene silencing of genes involved in flower pigment production to produce novel flower colours; gene silencing of genes involved in regulatory pathways controlling development or environmental responses to produce plants with novel growth habit or (for example) disease resistance; elimination of toxic secondary metabolites by gene silencing of genes required for toxin production.
A further aspect provides a process which includes introducing the vector into a plant, optionally including the further step of introducing a source of proteins encoded by TRV RNA1 into the plant.
A further aspect of the present invention provides a method which includes causing or allowing transcription from a construct as disclosed within the genome of a plant cell to produce a cytoplasmically-replicating RNA.
A further aspect of the present invention provides a method of reducing or suppressing or lowering the level of a target gene in a plant cell, the method including causing or allowing transcription from a vector as disclosed above.
In preferred forms the present invention is concerned with providing VIGS-based methods are useful in functional genomics. Thus in one aspect of the present invention, the target gene may be of unknown phenotype, in which case the VIGS system may be employed to analyse the phenotype by generating a widespread null (or nearly null) phenotype. The target gene may be essential, which is to say that the null phenotype is lethal to the cell or tissue in question.
This aspect of the invention may comprise a method of characterising a target gene comprising the steps of:
(a) silencing the target gene in a part or at a certain development stage of the plant using the TRV VIGS system described above,
(b) observing the phenotype of the part of the plant in which or when the target gene has been silenced.
Generally the observation will be contrasted with a plant wherein the target gene is being expressed in order to characterise (i.e. establish one or more phenotypic characteristics of) the gene.
The advantage of the TRV system over certain prior art constructs is discussed above. There are also several advantages of the current method over alternative methods in which the targeted gene is inactivated by insertional or other mutagenic procedures. The advantage over mutagenic procedures applies when there is more than one homologous gene carrying out the role of the target gene. Mutagenic procedures will not normally reveal a phenotype in that situation. A second situation where the current invention has advantage over both mutagenic and unregulated gene silencing procedures applies when the target gene has a lethal phenotype. The controllable attribute of the gene silencing will allow the phenotype of such genes to be investigated and exploited more efficiently than using the alternative methods available prior to the disclosure of the current invention.
Nor, for the identification of endogenous genes, would it be necessary to try and generate a transgenic plant in which gene silencing is already activated to observe the effect.
In a further aspect there is disclosed a method of altering the phenotype of a plant comprising use of the silencing method discussed above. Traits for which it may be desirable to change the phenotype include the following: colour; disease or pest resistance; ripening potential; male sterility.
In a further aspect of the present invention there is disclosed a virus or viral particle including, preferably encapsulating, a vector (or transcript from the expression cassette in the vector) according to the present invention.
In a further aspect of the present invention there is disclosed a kit comprising a vector as described above, plus a source of TRV RNA1 polypeptide or vector encoding the same (e.g. pBINTRA6).
In a further aspect of the present invention there is disclosed a host cell including a vector according to the present invention. These may be plant cells, or may be microbial (particularly bacterial and especially Agrobacterium) cells.
In a further aspect there is disclosed a plant, or plant tissue, including, or transiently transformed by, a vector of the present invention.
The invention will now be further described with reference to the following non-limiting Figures and Examples. Other embodiments of the invention will occur to those skilled in the art in the light of these.